Isocyanate, the essential cross-linking chemical for making polyurethane, are a major cause of occupational asthma world-wide. However, the mechanisms by which they cause disease remain unclear. We theorize that isocyanate asthma pathogenesis depends upon dynamic biochemical reactivity of isocyanate with multiple/different self-proteins/peptides, through metabolic pathways previously unappreciated for their connection to isocyanate allergenicity. Our specific hypothesis is that inhaled isocyanate reacts first, and preferentially, with the free thiol of the self-peptide, glutathione (GSH), which is preent at high concentrations (100-1000 M) in the airway fluid. However, thiol-linked isocyanate-GSH reaction products are quasi-stable at physiologic pH, and if not rapidly metabolized and excreted, can transfer the isocyanate group to another self-protein/peptide, especially albumin, one of the most abundant proteins in the body. Isocyanate conjugation of albumin is known to cause antigenic changes that trigger adaptive immunologic responses (specific IgE, T-cell responses) known to promote airway inflammation and asthma. The novelty of the present hypothesis vs. previously proposed mechanisms of isocyanate asthma is the formation of immunogenically distinct isocyanate antigens (isocyanate-albumin conjugates) via GSH-mediated transcarbomylation, rather than direct reactivity with isocyanate. If correct, this paradigm would explain many of the unique features of isocyanate vs. typical environmental asthma, and provide critical new insights into pathogenesis, risk factors, and diagnostic indicators of disease. In this grant application, we present unpublished Preliminary data that strongly support our novel hypotheses, and outline a Research Plan to better understand GSH-isocyanate reactivity and its connection with pathogenic responses to occupational exposure. Given the toxicity of isocyanates, the study design involves in vitro experiments followed by in vivo experiments in an animal model of isocyanate asthma, to directly test our hypotheses in a manner not possible in human subjects. The highly ambitious proposal will be facilitated through unique infrastructure (W.M. Keck Biotechnology Resource Laboratory) at the applicant's institution and collaboration with ongoing clinical isocyanate asthma research. The experimental data from the grant is expected to define key steps in isocyanate asthma pathogenesis, and biomarkers, upon which to base better strategies for disease surveillance and diagnosis. Successful completion of the proposed studies should also provide compelling evidence for further clinical studies to translate these initial findings into practical methods of disease prevention. The two specific aims are to Aim 1. Characterize the reaction products between glutathione (GSH) and isocyanate, and their carbamoylating capacity, under physiologic conditions in vitro. Aim 2. Determine the role of GSH-isocyanate interactions in the development of asthma in vivo in an animal model.